Method for the isolation and/or testing of genes and promoters involved in plant-nematode interactions using plants of the genus arabidopsis

ABSTRACT

The invention provides a method for infecting a root of a plant of the genus Arabidopsis with a plant parasitic nematode under monoxenic conditions, comprising the steps of: (a) contacting the root with a plant parasitic nematode in the presence of a gelling matrix dissolved in a nutrient medium that is substantially free of nematode inhibiting substances, and (b) allowing the nematode to infect the root by forming a feeding structure.

TECHNICAL FIELD

This invention concerns a method for the culturing of nematodes onplants. The method finds specific use in the screening and isolation ofnematode resistance genes and feeding-structure-specific genes andpromoters.

BACKGROUND AND RELEVANT LITERATURE

Plant-parasitic nematodes worldwide cause diseases of nearly all cropplants of economic importance with estimated losses of about $5.8billion/yr in the Unites States alone (Sasser and Freckman, 1987). Whilein tropical regions losses caused by nematodes are due mainly toroot-knot nematodes (Meloidogyne), in Europe cyst nematodes of thegenera Globodera and Heterodera are regarded as serious pests andimportant limiting factors in potato, rapeseed and sugarbeetcultivation, respectively. An increasing amount of crop damage is alsobeing ascribed to free-living nematodes (e.g. Pratylenchus ssp). Only asmall number of resistant crop varieties have emerged from breedingprogrammes for e.g. potato, sugarbeet, tomato, soybean and oil radish(Dropkin, 1988).

Through coevolution of plants and pathogens, plants have developeddefense mechanisms against their pathogens. For successful infection ofa plant, it is essential that the pathogen circumvents or suppresses thedefense mechanism of the plant. In genetical terms, specificplant-pathogen interactions can be described by the gene-for-gene model.In this model an elicitor molecule (e), encoded by an avirulence gene(E) from the pathogen, interacts with a receptor molecule (r), encodedby a resistance gene (R) from the plant, which switches on the defensemechanism. Phenotypically, this mechanism becomes visible through thehypersensitive response (HR): local death of host cells around the siteof infection which inhibits further development of the pathogen. Thegenetics of such gene-for-gene relationships are well documented forbacterial and fungal pathogens (Gabriel and Rolfe, 1990). Recent datafrom Whalen et al. (1991) indicate a degree of homology betweenresistance genes from Arabidopsis and soybean although these species arenot related. The gene-for-gene model has been suggested forplant-nematode interactions (Jones et al. 1981; Turner et al., 1983).Furthermore, the hypersensitive response that is observed in cultivarsthat are resistant against a particular nematode species and for whichthe resistance has been mapped to a single dominant locus (Rick andFobes, 1974; Delleart and Meijer, 1986; Omwega et al. 1990), indicatesthat gene-for-gene relationships may also function in plant-nematodeinteractions. The presence in soil of a resistance against nematodes ina particular variety persists under monoxenic culture conditions (e.g.Muller, 1978; Paul et al. 1990, Sanft and Wyss, 1990). Dominantresistance genes are being mapped in some of these varieties (e.g.potato, Barone et al., 1990; tomato, Williamson, 1990) but thecomplexity of the genomes of these crop plants as well as the complexityof the plant-nematode interaction under laboratory conditions hasprevented isolation of such genes to the present day. Most of theprogress in the techniques of plant-gene isolation with the help ofRestriction Fragment Length Polymorphism (RFLP)-mapping and chromosomewalking (Bleecker et al. 1991), T-DNA insertion mutagenesis (Feldmann,1991) or transposon-mutagenesis (Altmann et al. 1991) is accomplishedwith a small crucifer Arabidopsis thaliana. The genome size, the shortgeneration time and the well developed classical genetics for thisspecies are at the basis of this progress. One important factor for theisolation of a gene of interest is the ability to screen phenotypicallyfor the dominant presence of the gene in crossings with lines that arerecessive for that trait. One major drawback of Arabidopsis in theidentification of resistance genes is the limited number ofplant-pathogens that are able to infect on this species. Known examplesare now limited to a few bacteria (Simpson and Johnson, 1990; Whalen etal., 1991) and fungi (Koch and Slusarenko, 1990). Successful infectionof Arabidopsis with plant-parasitic nematodes, both in soil or undermonoxenic conditions, has never been reported in the literature, despitethe fact that nematodes can be major pests in agronomically importantcruciferous plants as sugarbeet and rapeseed. Standard conditions usedin nematology and plant tissue culture does not lead to successfulinfection of nematodes on Arabidopsis in soil or in tissue culture(unpublished results, MOGEN, Leiden, NL; Dept. Phytopathology, Univ.Kiel, Germany; Rothamsted Expt. Station, Harpenden, UK).

DESCRIPTION OF THE FIGURES

FIG. 1. Effects of Agar type on infection of Heterodera schachtii onArabidopsis thaliana. The inoculation with nematodes was done 10 d aftergermination. The medium used to dissolve the different agars was optimalmedium according to this invention. Results are expressed as apercentage of the mean number of infections in Daichin agar (n=45).DA=Daichin Agar, BA=Difco Bacto agar, TGA=technical grade agar,GR=Gelrite. All agar concentrations were 0.8% (w/v). Gelrite was at0.25% because of its higher water-retaining capacity.

FIG. 2. Medium effects on infection of Arabidopsis thaliana typeLandsberg erecta with Heterodera schachtii. Mean number of females/plantgrown in different media (n=45); 0.5 MS=half strength Murashige andSkoog (1962) medium, DB=Dropkin and Boone (1966) medium, Redei (1965)medium, Sijmons=medium according to this invention.

FIG. 3. Effect of Daichin agar concentration on infection of Arabidopsisthaliana type Landsberg erecta with Heterodera schachtii. The medium wasSijmons medium according to this invention. Mean number of successfulinfections per plant (n=45).

FIG. 4. Effect of sucrose concentration on infection of Arabidopsisthaliana type Landsberg erecta with Heterodera schachtii. The medium wasSijmons medium according to this invention. Mean number of successfulinfections per plant (n=45).

FIG. 5. Binary vector pMOG452. This plasmid is a derivative of pMOG23and contains a promoterless GUS construct to `fish` for promoters thatcan be used for expression of genes in a nematode feeding structure.

FIG. 6. Restriction map of fragment of Ti-plasmid pTiB6 used for theconstruction of a disarmed helper plasmid.

FIG. 7. Intermediate vector pMOG579, used to create the disarmed helperof Agrobacterium strain MOG101 which was used for Arabidopsistransformation.

SUMMARY OF THE INVENTION

The invention provides a method for infecting a root of a plant of thegenus Arabidopsis with a plant parasitic nematode under monoxenicconditions, comprising the steps of:

(a) contacting the root with a plant parasitic nematode in the presenceof a highly purified gelling matrix dissolved in a nutrient medium thatis devoid of nematode inhibiting substances,

(b) allowing the nematode to infect the root by forming a feedingstructure.

Another embodiment of the invention is a method for the screening of anecotype or a mutant of Arabidopsis for its relative resistance to aplant parasitic nematode, comprising the steps of:

(a) infecting the ecotype with a plant parasitic nematode using a methodas above, and

(b) counting the number of nematode feeding structures formed in theroots of said ecotype.

A further preferred embodiment of the invention is a monoxenic plantroot system of a plant of the genus Arabidopsis, comprising a rootsupporting substance in a suitable nutrient medium which is devoid ofnematode inhibiting substances, and a root which is infected with aplant parasitic nematode, whereby said root has at least one nematodefeeding structure. A more preferred embodiment comprises a monoxenicplant root system, wherein the root supporting substance is a highlypurified gelling matrix. A still further preferred embodiment is amonoxenic plant root system according to the invention, wherein saidhighly purified gelling matrix comprises 0.8% Daichin agar solution in anutrient medium, more preferably, wherein the medium contains 1%sucrose.

The invention finds specific use in a method for the isolation of a geneinvolved in resistance to a plant parasitic nematode from an ecotype ofArabidopsis, comprising the steps of:

(a) selecting an ecotype with a relatively high resistance to said plantparasitic nematode identified using any one of the methods above, and

(b) crossing said relatively resistant ecotype with a different ecotypewhich is relatively susceptible to infection against said plantparasitic nematode and which has a defined genetic background in termsof screenable genetic markers,

(c) linking the said gene involved in resistance to the plant parasiticnematode to one of said screenable markers through a series ofback-crossings of the most resistant progeny of said crossings with saidrelatively susceptible ecotype, until a sufficiently close linkage ofsaid gene with said screenable markers has been obtained to isolate thegene involved with resistance,

(d) isolating the said gene involved in resistance to said plantparasitic nematode on the basis of said linkage.

Further embodiments of the invention comprise genes involved in relativeresistance to a plant parasitic nematode obtained using a method of thepreceding paragraph.

Yet another embodiment of the invention comprises a method for theisolation of a promoter that is capable of driving the expression of agene in a nematode feeding structure from a plant of the genusArabidopsis, comprising the steps of:

(a) transforming a plant cell of the genus Arabidopsis with arecombinant polynucleotide which contains a structural coding sequenceencoding a screenable or selectable marker which is devoid of a promoterthat is active in a plant,

(b) generating a whole transformed plant from the cell transformed instep (a),

(c) infecting the roots of said plant obtained in step (b) with a plantparasitic nematode capable of causing a feeding structure in said rootsusing a method according to any one of the methods above,

(d) selecting plants the roots of which have feeding structures,

(e) selecting a plant root wherein the selectable or screenable markeris present in said feeding structure, and

(f) isolating the promoter that is upstream of the structural codingsequence encoding the selectable or screenable marker, causing theexpression of said structural coding sequence, from the genome of theplant selected in step (e). The invention also comprises a promoterobtained using the said method.

DETAILED DESCRIPTION OF THE INVENTION

It was found that a range of nematode species (among which the cystnematodes Heterodera schachtii, H. trifolii, H. cajani, the root-knotnematodes Meloidogyne incognita and M. arenaria and the migratorynematode Pratylenchus penetrans) can be grown on roots of Arabidopsisthaliana under monoxenic conditions if the medium at least comprises aroot supporting substance, which preferably comprises a highly purifiedgelling matrix (such as a tissue-culture-quality agar) that meets thefollowing requirements; a) the matrix has sufficient water-retainingcapacity but provides enough mechanical support for invasion ofinfective nematode juveniles into roots of Arabidopsis, it is able todissolve in used nutrient solutions at physiological pH, autoclavableprior to gelling and preferably transparent for routine analysis ofinfected root systems, b) a nutrient solution that is optimized forhydroponic root culture in combination with plant parasitic nematodes.c) and essentially void of any nematode-inhibiting substances. Apreferred gelling matrix comprises a tissue-culture-quality agar (e.g.Daichin agar) dissolved in a suitable nutrient medium. A suitable mediumcan be selected from known tissue culture media such as Murashige &Skoog medium (1962), or media used for nematode cultures on plant roots(Dropkin & Boone, 1966). Especially good results can be obtained byusing a medium containing: 2.5 mM K⁺, 1.27 mM Ca²⁺, 0.2 mM Mg²⁺, 2.54 mMNO₃ ⁻,0.5 mM H₂ PO₄ ⁻, 0.2 mM SO₄ ²⁻, 2 μM Na²⁺, 1.8 μM Mn²⁺, 0.14 μMZn²⁺, 60 nM Cu²⁺, 24 nM Co²⁺, 24 μM Cl⁻, 9 μM BO₃ ³⁻, 60 nM MoO₄ ²⁻, 20μM Fe³⁺ NaEDTA, pH 6.4, 1% (w/v) sucrose and 0.8% (w/v) Daichin agar,hereinafter referred to as the Sijmons medium. Further optimizations ofculture media can be done to improve the infection rate of a particularnematode-Arabidopsis interaction, which are within the scope of thisinvention. All the mentioned nematode species showed multiple infectionson single root systems in the said medium and were able to make completelife cycles while parasitizing Arabidopsis roots. Juveniles of Globoderarostochiensis were able to infect the roots under these monoxenicconditions but a strong necrosis at the site of infection preventedfurther development of the nematode. Juveniles of Heteroderagoettingiana were strongly stimulated by Arabidopsis roots in agar butthe roots were destroyed at the site of invasion and no developingjuveniles could be observed.

A preliminary screen of 74 different ecotypes of Arabidopsis forresistance against H. schachtii resulted in a range of infection rates(between 1.7 and 11.1 females/plant). This indicates that the geneticbackground of the Arabidopsis ecotype does influence the pathogenicityof the nematode. Hence we predict the possibility to identify andisolate dominant resistance genes from this plant using the techniquesfrom the present state of the art in molecular biology. The number ofsuccessful infections can be scored easily (without staining ordestructive root analysis) under monoxenic conditions. This eliminatesthe use of soil during screening and further adds to the potential ofthe monoxenic culture system. Plants that score favourably when assayedfor nematode resistance can immediately be brought to floweringconditions for seed harvest. Feeding-structures that develop inside theroots can be seen at low magnification and are easy to isolate with aminimum of contaminating cells. These feeding-structures are anexcellent source for RNA's that are specifically expressed in thesestructures. This allows the isolation, preferably using molecularenrichment procedures (Dickinson et al., 1991) of genes corresponding tothese RNA's. Using an (anti)sense approach, where the (anti)sense geneis directed against an essential gene for the development or maintenanceof feeding structures can be specifically inhibited. Alternatively, thepromoters that are associated with such essential genes can be used todrive the expression of genes inhibiting normal feeding-structuredevelopment or a normal feeding behaviour of the nematode.

The development of culture conditions for obligate nematodes on ecotypesof Arabidopsis thaliana offers formerly unknown possibilities to developplants with increased resistance against plant parasitic nematodes.

The following aspects are outlined in some detail for purposes ofillustration only and are not intended to limit the scope of thisinvention, as a skilled person may design alternative approaches.

I A first typical approach for obtaining nematode resistance genes,which can be introduced into economically important crop plants in orderto increase their resistance against plant-parasitic nematodes, willgenerally involve the following steps,

1) Screening of Arabidopsis ecotypes for resistance against nematodes byinfecting roots with a compatible (i.e. infectious) nematode using aninfection method according to the invention, selecting a resistant plantline, and preferably making the ecotype isogenic through repeatedbackcrosses,

2) Mapping the gene or genes determining the resistance through crossesof the (isogenic) resistant line with Arabidopsis thaliana ecotypeColumbia or Landsberg erecta, or other ecotypes which have well-definedgenetic backgrounds and are susceptible to the nematode, again using themedium conditions according to the invention, for the screening ofnematode resistance of the offspring,

3) Isolate the mapped nematode resistance (NR) gene from the saidresistant ecotype, by using gene tagging methods such as T-DNA insertionmutagenesis (Feldmann, 1991), transposon-tagging (Altmann et al. 1991)or RFLP mapping followed by chromosome walking (Bleecker et al., 1991).In the first method, Agrobacterium is used to perform saturationmutagenesis through the insertion of T-DNA's after which the transformedpopulation is screened for mutants in the resistance gene. Probing withT-DNA fragments and inverted PCR amplification allows the isolation ofthe flanking sequences. These in turn make it possible to clonesurrounding DNA fragments that can be re-transformed into the mutantline, allowing a phenotypic selection of clones containing the NR-gene.For the second method, it has been shown that transposons, such as Actransposase, also work in heterologous plant species such as tomato(Dickinson, 1991) or Arabidopsis (Altmann et al., 1991). A transgenicplant line is selected carrying an Ac element that is closely linked tothe NR-gene (by RFLP mapping) which can then be used for transposonmutagenesis of the NR-gene. Probing with Ac sequences and inverted-PCRamplification allows the isolation of flanking sequences followed by thesteps described above for the first method. The third method involvesRFLP-mapping followed by chromosome walking in order to identify andisolate the NR-gene (Bleecker, 1991).

4) Identify and isolate a promoter, e.g. through promoter analysis withmarker genes such as glucuronidase, that is functional in the plant tobe protected at the site of infection;

5) Cloning the said NR-gene behind the promoter, in an expressioncassette. In many cases also the entire resistance gene, with its ownpromoter, can be used directly after cloning in an expression cassette.

6) Introduce the expression cassette into plant material from which newplants can be generated.

7) Generate whole new plants from the transformed plant material.

Subsequently, transformed plants on which nematodes can be cultured canbe assayed for nematode resistance through growth of the plants in soilinfected with nematodes or under monoxenic conditions using an infectionmethod according to this invention.

II A second typical approach for obtaining nematode resistance gene,which can be introduced into economically important crop plants in orderto increase their resistance against plant-parasitic nematodes, willgenerally involve the following steps,

1) screening of Arabidopsis ecotypes or mutants of a resistant ecotypefor susceptibility against nematodes by infecting roots with anincompatible (i.e. non-infectious) nematode (e.g. Globoderarostochiensis or H. goettingiana) using an infection method according tothe invention, selecting a susceptible plant line, and preferably makingthe ecotype isogenic through repeated backcrosses,

2) mapping the resistance gene from e.g. Arabidopsis thaliana ecotypeColumbia or Landsberg erecta through crosses with the (isogenic)susceptible ecotype or mutant, again using the medium conditionsaccording to the invention,

3) to 7) as described for the first approach.

It should be understood that for the purposes of this description theterm "resistance gene" is not limited to genes involved in recognitionreactions as described by the gene-for-gene model. In mutant screenswith a compatible nematode, as described by the first approach, mutatedgenes may be encountered that are essential for steps after the initialrecognition phase such as feeding-structure induction or maintenance. Asmutations in such essential genes, hereinafter referred to as `EssentialGenes` will give phenotypical resistance in the culture conditions asdescribed in this invention, the genes carrying such a mutation can beidentified.

III the following steps will illustrate a third typical approach forobtaining nematode resistance genes of the latter category, which can beintroduced into economically important crop plants in order to increasetheir resistance against plant-parasitic nematodes,

1) Identify mutations in Essential Genes that are essential for any stepin the pathogenicity of parasitic nematodes by screening for resistancein mutants of a susceptible ecotype of Arabidopsis by infecting rootswith a compatible nematode, using a method according to the invention.

2) mapping the resistance gene through crosses with e.g. Arabidopsisthaliana ecotype Columbia or Landsberg erecta,

3) isolating the wild type equivalent of the mutant gene (if mutation isrecessive) or the mutant gene directly (if the mutation is dominant)from the said mutant, including its promoter, by complementationexperiments as e.g. described in step 3 of approach I.

4) cloning the said wild-type gene in an antisense direction behindasuitable promoter, and then steps 5)-7) as described for the firstapproach with the proviso that the NR-gene is in the antisense directionwith respect to the promoter.

Alternatively, one can also make use of the `sense` approach (Napoli etal. 1990; Van der Krol et al. 1990) in which in special cases theactivity of the endogenous gene is suppressed by expression of theincoming homologous gene.

In this approach (III), the NR-gene is in fact an (intact)`Essential-Gene` which is overexpressed, but as a result of theinhibitory effect resulting from the overexpression, the `EssentialGene` becomes a nematode resistance gene according to the invention.

IV Alternatively, resistance genes as isolated from Arabidopsis in thethird approach described above, can be used as probes to isolatehomologous genes and their particular promoter from other plant speciesthat are of agronomic importance.

V Alternatively, the culturing method provides a way for easy enrichmentof feeding-structure tissue, without the need of synchronized infectionor dissecting said structure from the roots. The feeding-structures thatdevelop inside the roots of Arabidopsis obtain several times the size ofthe surrounding epidermal and parenchymal tissue and are therefore easyto isolate with a minimum of contaminating cells, even at lowmagnification. Messenger-RNA isolated from such an enriched tissuesample can be used for the development and screening of substractioncDNA libraries (Dickinson et al., 1991), for the identification of genesthat are specifically expressed at this stage of infection. Through thisprocedure, genes that are specific for feeding-structure tissue andpossibly essential for the induction or maintenance of thefeeding-structure can be isolated and used in an (anti)sense approachthat is similar to that described above in approach III.

VI The root system according to the invention can be conveniently usedfor the isolation of promoters that are capable of expressing genes inthe feedig structures, prefereably resistance genes. Such promoters areisolated, for instance via interposon tagging (Topping et al., 1991,Developm. 112, 1009-1019; Koncz, C. et al., (1989) Proc. Nat. Acad. ofSci. U.S.A. 86, 8467-8471). The random integration of the T-DNA enablesthe identification of promoter sequences that are active in the feedingstructures. This type of interposon tagging of promoter sequences isespecially well established in Arabidopsis (Kertbundit et al., 1991,Proc. Nat. Acad. Sci. USA 88, 5212-5216) and tobacco (Topping et al.,1991, Developm. 112, 1009-1019). The tagged promoter sequences upstreamof the GUS structural coding sequence can for instance be isolated withinverted polymerase chain reaction (Does et al. 1991, Plant Mol. Biol.17, 151-153). Once suitable regulatory sequences are identified or genesthat are transcribed inside the feeding structure, they can be used asprobes for the isolation of homologous sequences from other plantspecies.

The following examples further illustrate the invention.

EXPERIMENTAL

The methods described in this section for maintenance and sterilizationof nematode species are routine procedures in the art of Nematology anddo not part of the invention.

Cultivation of Plants

Arabidopsis seeds were soaked 2 min in 70% EtOH and surface sterilizedeither for 5 min in 0.8% Ca(OCl₂), 0.05% Tween 20 or for 8 min in 5%Ca(OCl₂), 0.05% Tween 20, depending on the degree of contamination ofthe seeds. Sterilized seeds were washed at least 3 times in sterilewater and transferred to agar containing growth media.

The optimal medium for monoxenic development of nematodes contained: 2.5mM K⁺, 1.27 mM Ca²⁺, 0.2 mM Mg²⁺, 2.54 mM NO₃ ⁻, 0.5 mM H₂ PO₄ ⁻, 0.2 mMSO₄ ²⁻, 2 μM Na ²⁺, 1.8 μM Mn²⁺, 0.14 μM Zn²⁺, 60 nM Cu²⁺, 24 nM Co²⁺,24 μM Cl⁻, 9 μM BO₃ ³⁺, 60 nM MoO₄ ²⁻. Fe was added as 20 μM Fe³⁺NaEDTA. The pH was adjusted to 6.4 with 1 N KOH. Just prior tosterilization (20 min 110° C.), 1% (w/v) sucrose and 0.8% (w/v) Daichinagar (Brunschwig Chemie BV, POB 70213, Amsterdam, The Netherlands) wasadded. Both 9 cm Petri dishes with 15 seeds or 24-well tissue cultureplates (Greiner, Germany) with 2 seeds per well were used to study mediaconditions. Ecotype screening was done in the 24-well plates. Fortesting of different nematode species, seeds were arranged in a row onthe top half of a Petri dish. After a 3 d germination period, the plateswere slightly tilted to let the roots grow downwards. The plates weresealed with Parafilm and kept at 23°-25° C. with 16 hr L/8 hr D.

Maintenance of Nematode Populations.

The different nematode species used in these studies were eithermaintained in pot cultures or sampled from field populations on theirrespective hosts as indicated in Table 1. Monoxenic stock cultures of H.schachtii were maintained in vitro on Sinapis alba cv. Albatros for 6weeks and stored at 4° C. until use (Grundler, 1989).

Sterilization of Nematode Species

Egg-suspensions were prepared from crushed fresh cysts or hand-pickedegg masses in the case of Meloidogyne. The suspension was placed on asterile Swinnex disc filter holder with a cellulose nitrate membrane(Schleicher & Schuell, pore size 5 μm) or on 20 μm nylon gauze fixed ina plastic ring. The eggs on the filter were exposed to 0.1% HgCl₂ for 4min and washed 4 times with 5 ml sterile water (Grundler, 1989). Thesterile eggs were scooped off the membrane onto the agar surface.

Deposit of microorganisms

Binary vector pMOG23 has been deposited in E. coli K-12 strain DH5α,deposited at the Centraal Bureau voor Schimmel-cultures on Jan. 29, 1990under accession number CBS 102.90).

EXAMPLE 1

Culture conditions for infection of Arabidopsis

Aseptic cysts of H. schachtii containing J₂ were either cut opendirectly in the vicinity of growing root tips, or transferred on a 200μm mesh plastic sieve and placed in a sealed glass funnel containing a 3mM ZnCl₂ hatching stimulant solution. After 3 d dark incubation (25°C.), hatched J₂ could be harvested, washed 3 times with sterile waterand suspended in 0.5% Gelrite for reproducible inoculations.Inoculations were done 7-10 days after sowing, either with crushed cystsor with 25-70 juveniles per plant, depending on the type of experiment.All procedures were performed under aseptic conditions on a minimum of45 plants.

The effects of different agar qualities on the number of successfulinfections in monoxenic culture are illustrated in FIG. 1. The effectsof different media, sucrose concentration and Daichin agar concentrationon the number of developing females per plant or the number ofsuccessful infections per plant are illustrated in FIG. 2. The followingArabidopsis ecotypes were tested: An-1, Ba-1, Bch-1, Bl-1, Bla-1, Bn-0,Bor-0, Cal-0, Chi-0, Ci-0, Co-4, Col-0, Ct-1, Cvi-0, Edi-0, En-2, Esc-0,Est-0, For-1, Gd-1, Go-0, Gre-0, Ha-0, Hl-0, Hm-0, Hs-0, Ita-0, Ka-0,Kil-0, Kin-0, Kl-0, Kr-0, La-0, La-er, Lan-0, Lc-0, Ll-0, Map-0, Mc-0,Mh-0, Mr-0, Ms-0, Old-2, Ove-0, Pa-3, Per-1, Pi-0, Pla-0, Po-0, 0, Pt-0,Rou-0, Rsch-0, Sac-0, Sah-0, Se-0, Sei-0, Set-0, Sf-0, Sr-0, Stw-0,Su-0, Sue-0, Sy-0, Ts-1, Tu-0, Tul-0, Ty-0, Wa-1, Wc-1, Wil-3, Ws-0,Wt-1, Yo-0, Ze-0. The codes are according to the population codes of theAIS-Seed bank listings (Kranz and Kirchheim, 1987). The most resistantecotypes (i.e. the lowest mean number of developing females per plant)were Sah-0, Lan-0, and Kil-0. The most susceptible ecotypes were Gre-0and La-0. Presently Sah-0 and Lan-0 are the best candidates for mappingresistance genes. We predict that upon further screening of ecotypesmore, putatively even absolutely, resistant ecotypes will be found,which will be most preferred for use in mapping NR-genes.

Nematode species tested on Arabidopsis

The different nematode species that were tested for infectivity andcompletion of lite cycles are mentioned in Table 1. Except for G.rostochiensis and H. goettingiana, all other nematode species mentionedin Table 1 did have complete life cycles on Arabidopsis roots usingmedium according to the invention. The medium was optimized for H.schachtii and need not be the most optimal for the other species tested.Specific ion- and sucrose-concentrations, buffer types and gellingmatrices may be optimized further according to specific requirements ofa plant-parasitic nematode species. The nematode species mentioned inTable 1 are offered by way of illustration and not by way of limitation.

EXAMPLE 2

Construction of a promoterless GUS gene construct

The gene coding for GUS fused to a 3'nos terminator sequence but withoutany 5' regulatory promoter sequences was cloned as a EcoRI-BamHIfragment from pBI101 plasmid (Jefferson, 1987, Plant Mol. Biol. Reporter5, 387-405) into the multiple cloning site of binary vector pMOG23,resulting in binary plasmid pMOG452 (FIG. 5). pMOG452 was introducedinto Agrobacterium tumefaciens strain MOG101 by triparental mating fromE.coli, using HB101 pRK2013) as a helper. Transconjugants were selectedfor resistance to rifampicin (20 mg/l) and kanamycin (100 mg/l).

EXAMPLE 3

Construction of Agrobacterium strain MOG101

A binary vector system was used to transfer gene constructs intoArabidopsis plants. The helper plasmid conferring the Agrobacteriumtumefaciens virulence functions was derived from the octopine Ti-plasmidpTiB6. MOG101 is a Agrobacterium tumefaciens strain carrying anon-oncogenic Ti-plasmid (Koekman et al. 1982, Plasmid 7, 119-132) fromwhich the entire T-region was deleted and substituted by a bacterialSpectinomycin resistance marker from transposon Tn 1831 (Hooykaas etal., 1980 Plasmid 4, 64-75).

The Ti-plasmid pTiB6 contains two adjacent T-regions, TL (T-left) and TR(T-right). To obtain a derivative lacking the TL- and TR-regions, weconstructed intermediate vector pMOG579. Plasmid pMOG579 is a pBR322derivative, which contains the 2 Ti-plasmid fragments that are locatedto the left and right, outside the T-regions.(FIG. 6). The 2 fragments(shown in dark) are separated in pMOG579 by a 2.5 kb BamHI-HindIIIfragment from transposon Tn1831 (Hooykaas et al., 1980 Plasmid 4, 64-75)carrying the spectinomycin resistance marker (FIG. 7). The plasmid wasintroduced into Agrobacterium tumefaciens strain LBA1010 C58-C9 (pTiB6)which is a cured C58 strain in which pTiB6 was introduced (Koekman etal. (1982), supra), by triparental mating from E.coli, using HB101pRK2013) as a helper. Transconjugants were selected for resistance toRifampicin (20 mg/l) and spectinomycin (250 mg/l). A doublerecombination between pMOG579 and pTiB6 resulted in loss ofcarbenicillin resistance (the pBR322 marker) and deletion of the entireT-region. Of 5000 spectinomycin resistant transconjugants replica platedonto carbenicillin (100 mg/l) 2 were found sensitive. Southern analysisshowed that a double crossing over event had deleted the entire T-region(not shown). The resulting strain was called MOG101.

EXAMPLE 4

Transformation of Arabidopsis with pMOG452

Arabidopsis is transformed with Agrobacterium strain MOG101 containingthe binary vector pMOG452. Transformation is carried out usingco-cultivation of Arabidopsis thaliana (ecotype C24) root segments asdescribed by Valvekens et al. (1988, Proc. Nat. Acad. Sci. USA 85,5536-5540). Transgenic plants are regenerated from shoots that grow onselection medium (50 mg/l kanamycin), rooted and transferred togermination medium or soil.

Transgenic plant lines (T2 or later generations) are used for infectionwith plant parasitic nematodes and subsequently used for GUS analysis inthe feeding structures. The GUS assay in the feeding structures can becarried out substantially the same way as described for plant leaves(Jefferson, 1987, Plant Mol. Biol. Reporter 5, 387-405). The dissectedroots containing a feeding structure are assayed while still inside theagar; alternatively the agar can be removed before staining.

The results from such a screening experiment after inoculation with H.schachtii showed a surprisingly high number of plants with GUS activityinside the feeding structure and low or no GUS activity in other plantparts, thus indicating that regulatory sequences that drive geneexpression inside the feeding structure are tagged by the promoterlessGUS gene construct. One in 13 independent transgenic plant linesindicated a tagged promoter that was active in the feeding structurecells. This high frequency illustrates the feasibility to tag, andsubsequently isolate a promoter that can suitably be used for theexpression of nematode resistance gene in a plant cell.

EXAMPLE 5

Isolation of promoter from transgenic plants expressing GUS inside thefeeding structure

Transgenic plant lines that express GUS activity inside the feedingstructure are selected and used for isolation of genomic DNA. Regulatorysequences upstream (5') of the integrated GUS gene is isolated usinginverted PCR (Does et al. 1991, Plant Mol. Biol. 17, 151-153) with theprimers 5'-CCAGACTGAATGCCCACAGGC-3' (SEQUENCE ID NO:1) and5'-GGTGACGCATGTCGCGCAAG-3' (SEQUENCE ID NO:2). The amplified fragment isused to screen a genomic library of Arabidopsis for the isolation of agenomic clone that contains a suitable promoter for expression of a geneinside the feeding structure.

Alternatively, the first 200 bp of the GUS gene are used to probe agenomic bank made from the selected plants.

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                                      TABLE 1                                     __________________________________________________________________________    Nematode species on Arabidopsis thaliana type Landsberg erecta in             monoxenic conditions.                                                         Nematode   Origin.sup.1                                                                            Last Host                                                                            Comments                                          __________________________________________________________________________    Heterodera schachtii                                                                     RES/Kiel populations                                                                    oilseed rape,                                                                        complete life cycle in ca. 6 weeks,                                    mustard                                                                              necrosis at invasion and feeding site.            H. trifolii                                                                              RES field population                                                                    clover complete life cycle in ca. 2 months.              H. goettingiana                                                                          RES field population                                                                    field beans                                                                          J.sub.2  strongly stimulated by roots,                                        destroy                                                                       roots at invasion site, no developing                                         juveniles observed.                               H. cajani  India, 1 yr at RES                                                                      cowpea complete life cycle in ca. 2 months.              Globodera rostochiensis                                                                  RES greenhouse                                                                          potato/Desiree                                                                       few attempts to invade, strong necrosis, no                                   further development.                              Melidogyne incognita                                                                     RES/Kiel populations                                                                    tomato/Pixie                                                                         complete life cycle in 4-5 weeks, little or                                   no necrosis during invasion, galling,                                         females not within gall.                          M. arenaria                                                                              NCSU, since 1983                                                                        tomato/Pixie                                                                         complete life cycle in ca. 6 weeks, little                   RES greenhouse   or no necrosis during invasion, round galls.      Pratylenchus penetrans                                                                   Kiel/RES  carrots, maize                                                                       life cycle in 4 weeks, good development;                                      eggs deposited inside and outside of roots,                                   necrosis.                                         __________________________________________________________________________     .sup.1 RES = Rothamsted Expt. Station, NCSU = North Carolina State            University                                                               

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: Yes                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCAGACTGAATGCCCACAGGC21                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: Yes                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GGTGACGCATGTCGCGCAAG20                                                        __________________________________________________________________________

We claim:
 1. A plant root system comprising a root of a plant of thegenus Arabidopsis, a root-supporting substance, a nutrient medium and aplant-parasitic nematode that causes at least one nematode feedingstructure in said root for supporting development of the nematode for acomplete life cycle, the root supporting substance and nutrient mediumbeing suitable for supporting development of the nematode on the rootfor said complete life cycle, said root comprising the at least onenematode feeding structure supporting the development of saidplant-parasitic nematode, said root supporting substance comprising ahighly purified gelling matrix that provides sufficient water-retainingcapacity and mechanical support for supporting said development, saidroot supporting substance being substantially devoid ofnematode-inhibiting substances.
 2. A plant root system according toclaim 1, wherein said plant is of the species Arabidopsis thaliana.
 3. Aplant root system according to claim 1, wherein said plant parasiticnematode is selected from Heterodera schachtii, Heterodera trifolii,Heterodera cajani, Meloidogyne incognita, Meloidogyne arenaria, andPratylenchus penetrans.
 4. A plant root system according to claim 1,wherein the root supporting substance comprises a tissue-culture gradegelling-matrix dissolved in said nutrient medium.
 5. A monoxenic plantroot system according to claim 4, wherein the root supporting substancecomprises 0.8% Daichin agar (w/v) in 2.5 mM K⁺, 1.27 mM Ca²⁺, 0.2 mMMg²⁺, 2.54 mM NO₃ ⁻, 0.5 mM H₂ PO₄ ⁻, 0.2 mM SO_(4hu) 2-, 2 μM Na²⁺, 1.8μM Mn²⁺, 0.14 μM Zn²⁺, 60 nM Cu²⁺, 24 nM Co²⁺, 24 μM Cl⁻, 9 μM BO₃ ³⁻,60 nM MoO₄ ²⁻, 20 μM Fe³⁺ NaEDTA, 1% (w/v) sucrose, pH 6.4.
 6. A plantroot system according to claim 1, wherein the root supporting substancecomprises 0.8% Daichin agar.
 7. A plant root system as claimed in claim1, wherein said nematode feeding structure comprises a DNA sequence withan open reading frame encoding a selectable or screenable marker.
 8. Aplant root system according to claim 7, wherein the open reading frameencoding the selectable or screenable marker is transcribed under thecontrol of a promoter that is naturally present in the genome of theplant and is expressed in said feeding structure.
 9. A plant root systemaccording to claim 7, wherein the open reading frame encodesbeta-glucuronidase.
 10. A plant root system according to claim 7,wherein said plant is of the species Arabidopsis thaliana.
 11. A plantroot system according to claim 7, wherein said plant-parasitic nematodeis a cyst nematode.
 12. A plant root system according to claim 11,wherein said cyst nematode is Heterodera schachtii.
 13. A plant rootsystem according to claim 7, wherein the root supporting substancecomprises a tissue-culture grade gelling-matrix dissolved in saidnutrient medium.
 14. A plant root system according to claim 7, whereinthe root supporting substance comprises 0.8% Daichin agar.
 15. A methodcomprising infecting a root of a plant of the genus Arabidopsis with aplant parasitic nematode for the production of at least one nematodefeeding structure on the root for supporting development of the nematodefor a complete life cycle by contacting the root with the plantparasitic nematode in the presence of a composition consistingessentially of a root supporting substance and a nutrient mediumsuitable for supporting the development of the nematode on the root forsaid complete life cycle, said root supporting substance comprising ahighly purified gelling matrix that provides sufficient water-retainingcapacity and mechanical support for supporting said development, saidroot supporting substance being substantially devoid ofnematode-inhibiting substances.
 16. A method as claimed in claim 15,wherein the root is from an ecotype or mutant of Arabidopsis and themethod further comprises screening the root for the presence of feedingstructures to calculate the number thereof, and determining a resistanceof the ecotype or mutant from the number of feeding structures.
 17. Aroot of a plant of the genus Arabidopsis made by the method of claim 15wherein the at least one nematode feeding structure comprises a DNAsequence with an open reading frame encoding a selectable or screenablemarker.
 18. A root according to claim 17, wherein said nematode feedingstructure is produced by a process comprising infecting the plant with acyst nematode.
 19. A root according to claim 18, wherein said cystnematode is Heterodera schachtii.
 20. A root according to claim 17,wherein the open reading frame encoding the selectable or screenablemarker is transcribed under the control of a promoter that is naturallypresent in the genome of the plant and is expressed in said feedingstructure whereby said selectable or screenable marker is detectablypresent in said feeding structure.
 21. A root according to claim 17,wherein the open reading frame encodes beta-glucuronidase.
 22. A rootaccording to claim 17, which is of the species Arabidopsis thaliana. 23.A plant root system according to claim 3 wherein the root supportingsubstance is an agar selected from the group consisting of Daichin Agarand Difco Bacto Agar.
 24. A method as claimed in claim 15 wherein theroot-supporting substance and nutrient medium are suitable forsupporting infection of the plant by more than one female nematode. 25.A method as claimed in claim 15 wherein the root-supporting substanceand nutrient medium are suitable for supporting multiple infections ofthe plant by nematodes.